The ubiquitin-proteasome system regulates p53-mediated transcription at p21waf1 promoter.

The ubiquitin (Ub)-proteasome system (UPS) promotes the proteasomal degradation of target proteins by decorating them with Ub labels. Emerging evidence indicates a role of UPS in regulating gene transcription. In this study, we provided evidence for the involvement of UPS in the transcriptional activation function of tumor suppressor p53. We showed that both ubiquitylation and proteasomal functions are required for efficient transcription mediated by p53. Disruption of transcription by actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimadazole or alpha-amanitin leads to accumulation of cellular p53 protein. Proteasome inhibition by MG132 increases the occupancy of p53 protein at p53-responsive p21(waf1) promoter. In addition, the Sug-1 component of 19S proteasome physically interacts with p53 in vitro and in vivo. Moreover, in response to ultraviolet-induced DNA damage, both the 19S proteasomal components, Sug1 and S1, are recruited to p21(waf1) promoter region in a kinetic pattern similar to that of p53. These results suggested that UPS positively regulates p53-mediated transcription at p21(waf1) promoter.

Nuclear-mitochondrial genomic profiling reveals a pattern of evolution in epithelial ovarian tumor stem cells.

Analyses of genome orthologs in cancer on the background of tumor heterogeneity, coupled with the recent identification that the tumor propagating capacity resides within a very small fraction of cells (the tumor stem cells-TSCs), has not been achieved. Here, we describe a strategy to explore genetic drift in the mitochondrial genome accompanying varying stem cell dynamics in epithelial ovarian cancer. A major and novel outcome is the identification of a specific mutant mitochondrial DNA profile associated with the TSC lineage that is drastically different from the germ line profile. This profile, however, is often camouflaged in the primary tumor, and sometimes may not be detected even after metastases, questioning the validity of whole tumor profiling towards determining individual prognosis. Continuing mutagenesis in subsets with a mutant mitochondrial genome could result in transformation through a cooperative effect with nuclear genes - a representative example in our study is a tumor suppressor gene viz. cAMP responsive element binding binding protein. This specific profile could be a critical predisposing step undertaken by a normal stem cell to overcome a tightly regulated mutation rate and DNA repair in its evolution towards tumorigenesis. Our findings suggest that varying stem cell dynamics and mutagenesis define TSC progression that may clinically translate into increasing tumor aggression with serious implications for prognosis.

Human homologue of yeast Rad23 protein A interacts with p300/cyclic AMP-responsive element binding (CREB)-binding protein to down-regulate transcriptional activity of p53.

The tumor suppressor protein p53 regulates various cellular responses to DNA damage and plays a significant role in DNA repair. The nuclear p300/cyclic AMP-responsive element binding (CREB)-binding protein (CBP) proteins act as coactivators in supporting the transcription function of p53. We examined the role of the human homologue of yeast Rad23 protein A (hHR23A), one of the two human homologues of the Saccharomyces cerevisiae nucleotide excision repair gene product Rad23, in the p300/CBP-associated regulation of p53 activity. Overexpression of wild-type hHR23A inhibits the p53 transcriptional activity and results in a decreased steady-state protein level of cellular p53. The inhibitory effect of hHR23A can be overcome by the concomitant expression of p300, CBP, and p300 segments harboring C/H1 domain and neutralized by the coexpression of HIV accessory protein Vpr, which binds COOH terminus of hHR23A/B. Additionally, hHR23A was shown to interact in vitro and in vivo with p300 segments harboring C/H1 domain. These studies provide evidence for the involvement of hHR23A in the regulation of p53 activity through p300/CBP. Although the precise direct role of hHR23 proteins in regulation of p53 and DNA repair remains to be elucidated, our data suggest that the interaction between hHR23A and p300/CBP has important implications in cross-talk between the p53 pathway and DNA repair.

Enhanced sensitivity to anti-benzo(a)pyrene-diol-epoxide DNA damage correlates with decreased global genomic repair attributable to abrogated p53 function in human cells.

DNA damage from exposure to environmental chemical carcinogens and failure of repair systems to eliminate these lesions from the genome are considered as the crucial initial steps in the development of various human malignancies. Many cellular proteins are known to play vital roles to overcome the effects of DNA damage. Among such proteins, p53 is known to respond to DNA damage by accumulating in the nucleus and inhibiting cell cycle progression to facilitate DNA repair and the maintenance of genomic stability. In this study, we have investigated the role of p53 protein in modulating nucleotide excision repair of anti-benzo-(a)pyrene-diol-epoxide (BPDE)-DNA adducts and related effects using human fibroblasts with normal (p53-WT) and altered p53 protein (p53Mut and p53-Null). Interestingly, irrespective of the presence or absence of p53, the anti-BPDE dose-dependent p21 protein induction response was qualitatively comparable in all of the three cell lines. However, cells with defective p53 function were deficient for the removal of anti-BPDE-DNA adducts from the overall genome compared to cells with wild-type p53 activity. Strand-specific repair analysis within the individual strands of the p53 gene revealed decreased repair of adducts from the nontranscribed strand in p53-Mut and p53-Null cells. However, the repair of the transcribed strand appeared to be identical in all of the three cell lines. Furthermore, p53-Mut and p53-Null cells were more sensitive than p53-WT cells and displayed increased levels of anti-BPDE-induced apoptosis. Thus, wild-type p53 is required for the efficient global genomic repair of anti-BPDE-induced DNA adducts from the overall genome, but not for transcription-coupled repair of actively transcribed genes. These findings indicate that inefficient DNA repair of potentially cytotoxic and mutagenic lesions from the nontranscribed strand due to the loss of p53, but not the loss of p21, function might be responsible for enhanced cytotoxicity and apoptosis in human cells upon DNA damage.

O6-methylguanine-DNA methyltransferase in human fetal tissues: fetal and maternal factors.

O6-Methylguanine methyltransferase (O6-MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race and of maternal smoking and drug usage. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin, and brain. Each fetal organ homogenate exhibited a 3- to 5-fold level of interindividual variation of O6-MT. There did not appear to be any significant differences of O6-MT as a function of fetal race and age and in the tissues obtained from mothers who smoked cigarettes during pregnancy. The fetal tissues obtained from an individual using phenobarbital exhibited 4-fold increases in O6-MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest a possible enhancement of human fetal O6-MT by certain xenobiotics, with little if any modulation by racial factors and maternal smoking habits.

Modulation of (+/-)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase.

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.05-1 microM (+/-)-anti-BPDE resulted in elevated p53 protein levels as compared to the constitutive levels of control cells. The rapid induction response, detectable within a few hours, was sustained up to a period of at least 24 h. Repair-proficient and repair-deficient (XPA) human lymphoblastoid cells showed a similar response. The poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3-AB), diminished the p53 induction response by concomitantly decreasing the extent of (+/-)-anti-BPDE induced DNA damage in cells pretreated with the inhibitor. However, the direct involvement of poly ADP-ribosylation was also apparent as 3-AB was able to attenuate (approximately 50%) the p53 response by post-damage inhibitor treatment of the cells. Inhibition of cellular DNA replication by hydroxyurea and AraC, in the presence or absence of DNA damage, also resulted in rapid p53 accumulation in repair-deficient cells. On the contrary, inhibition of protein kinase C (PKC) by calphostin-C led to an abrogation of (+/-)-anti-BPDE mediated p53 induction. Analysis of the downstream effects of carcinogen treatment showed that the lymphoblastoid cells undergo DNA fragmentation indicative of apoptosis while fibroblasts exhibit cell cycle arrest at the G1-S boundary.

Immunoanalysis of ultraviolet radiation induced DNA damage and repair within specific gene segments of plasmid DNA.

The region-specific heterogeneity of repairing DNA damage has been established in several biological systems. A flexible and sensitive approach, based upon DNA damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments. Membrane transblotted DNA restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers. Sensitivity of dimer immunodetection increases proportional to fragment concentration and size. Antibody binding was detectable in a 0.5 kb fragment obtained from approx. 100 ng of restriction digested phage lambda DNA irradiated with 50 J m-2. Dimers within larger fragments (greater than 5 kb) could be detected at ultraviolet doses as low as 1 to 2 J m-2. To determine the occurrence of preferential repair in prokaryotic cells, damage was assessed in DNA sequences established in various Escherichia coli strains. In vivo repair of 8.9 kb vector and 6.4 and 3.2 kb gene inserts occurred with an approximate t1/2 of 45 min in UvrABC excision repair-proficient strains. Antibody binding sites were retained by DNA within repair-deficient strains. Compared to UvrABC, the repair of DNA fragments mediated by T4 endonuclease V was rapid and complete within 30 min of cellular irradiation. The efficient repair in DenV+ strain is attributable to a highly processive repair enzyme rather than to selective repair of actively replicating target genes. The results demonstrate the exceptional ability of antibodies specific for altered biomolecular lesions to map damage and repair in gene segments episomally established within cells.

Quantitative analysis of carbodiimide modified DNA and immunoprobing by adduct specific antibodies.

Antibodies have been raised against N-cyclohexyl-N-(4-methylmorpholinium)ethyl carbodiimide (CMC) modified single-stranded DNA and characterized by competitive and non-competitive immunoassays to be highly specific for CMC base adduct in homopolymers poly(dG), poly(dT) and DNA. The antibodies recognize picogram concentrations of CMC treated DNA with no cross reactivity to at least 1000-fold excess of unmodified DNA or CMC treated poly(dA). The detection limit of antibodies at 1.4 fmol CMC adduct allows quantitation at a CMC/base ratio of 4.6.10(-7). Based upon single modified base-containing synthetic oligomers, a 7-fold higher binding preference is observed for CMC modified thymine than guanine bases. CMC binding to supercoiled DNA is found to depend upon reaction temperature and ionic strength. CMC-modified supercoiled SV40 and ColE1 DNA, exhibit specific antibody binding proportional to the DNA concentration and extent of CMC modification. However, antibody binding observed is independent of the conformation or strandedness of CMC-modified DNA. DNA extensively modified with CMC retains its inherent capacity to specifically and quantitatively hybridize with complementary DNA immobilized to membranes upon direct blotting or Southern transfers from gels. Hybridized CMC-DNA, through antibody binding, provides for the sensitive and non-isotopic detection of the target DNA sequences.

Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool.

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.

Site-specific gap-misrepair mutagenesis by O4-ethylthymine.

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.

Repair of base alkylation damage in targeted restriction endonuclease sequences of plasmid DNA.

Sequence specific ethylation damage and repair of ethyl-adducts in selected restriction endonuclease recognition sites within p220-ras plasmid DNA was assessed by a modified Southern blotting coupled immunoprobing technique. In situ UV irradiation of DNA in gels clearly ameliorated the immunodetection of minute amounts of facultative fragments generated due to inhibition of enzyme cleavage site by covalent alkylation modification of the cognate sites. Specific and quantitative localization of induced facultative fragments was achieved in as low as 1 ng of DNA digest corresponding to a peak intensity below 0.1 absorbance unit upon laser scanning. An ENU dose dependent increase in the intensity of representative 7.1 and 7.7 kb facultative fragments was observed as a result of cleavage block at EcoRI (G/ATTC) and BamHI (G/GATCC) restriction endonuclease sites, respectively. To determine the repair in prokaryotic cells, the half-life of repairable alkyl-adducts was assessed in plasmid DNA established in various Escherichia coli strains as a function of post-treatment incubation time in the recovery medium. The repair is indicated by the gradual disappearance of the 7.1, 7.7, 11.9 and 5.5 kb facultative fragments within the wild-type and mutant E. coli strains. The ethyl-adducts within EcoRI and BamHI restriction sites were effectively lost from the target DNA in repair-proficient E. coli with an estimated t1/2 of approximately 40 min. However, decreased overall rate and at least 2.2-times lesser extent of repair was observed in the repair-deficient (ada+ogt-) and (ada-ogt+) cells. No measurable repair was noticed in alkyltransferase defective double mutant (ada-ogt-) even after 2 h of post-treatment incubation. The repair of ethyl-adducts at NotI site (GC/GGCCGC) in 5.5 kb facultative fragment occurred at a relatively faster rate (t1/2 of 27 min) in wild-type bacteria. A 1.5-fold slower repair of ethyl-adducts in BamHI and EcoRI sequences containing G/G and A/G at their cleavage sites was observed compared to C/G in NotI sequence. These results demonstrate the regioselective induction of alkyl-adducts in ethylated DNA and their differential repair in E. coli due to varied efficiency of the repair enzymes for promutagenic DNA base lesions present in different sequence context.

Induction and processing of promutagenic O4-ethylthymine lesion in specific gene segments of plasmid DNA.

High affinity antibodies were used for the quantitative assessment of the miscoding O4-ethylthymine (O4-EtThy) base lesion in nanogram amounts of membrane transblotted restriction fragments of ENU treated DNA. The polyclonal antibody (TB3) specifically recognized attomoles of the alkylation adducts in modified DNA with no cross-reactivity to an excess of unmodified DNA. The sensitivity of the immuno-quantitative method was determined to be in the range of 76 attomoles to 2.43 fmol, corresponding to 0.24 x 10(-7) to 7.9 x 10(-7) adducts per nucleotide in plasmid DNA. Modification levels in ras and tk genes were estimated as 0.025 and 0.014 adducts respectively. Specific antibody binding was proportional to the dose of ENU and size of the DNA fragments. In differentially ethylated ras gene, the amount of O4-EtThy was quantified as 0.026, 0.08 and 0.13 adducts per gene fragment. A DNA concentration dependent antibody binding was observed with large (23.13 and 9.41 kb) and smaller (2.02 kb) fragments of HindIII digested ENU treated phage lambda DNA. To monitor the repair of O4-EtThy lesions in specific segments, damage was assessed in sequences of plasmid DNA established in various Escherichia coli strains. The loss of antibody binding to O4-EtThy adducts in ethylated DNA fragments of 6.4 kb ras gene and 3.6 kb tk gene occurred with an approximate t1/2 of 45 and 35 min, respectively, in the repair proficient wild type E. coli. On the contrary, no repair was seen in the alkyltransferase deficient double mutant ada-ogt- strain. The results specifically demonstrate the sensitivity of the immunological technique and the unique ability of the O4-EtThy specific antibodies to scan this promutagenic base lesion and its repair in very small amounts of selected gene segments in DNA.

Repair analysis of promutagenic (+)-anti-BPDE DNA adduct in transcriptionally active sequences of plasmid DNA in Escherichia coli.

The extent of formation and repair of promutagenic (+)-anti-BPDE-N2-dG in transcriptionally active thymidine kinase (tk) gene insert and vector DNA fragments was assessed in the (+)-anti-BPDE treated plasmid p220-tk within the Escherichia coli hosts of varying repair potential. Polyclonal antibody (BP1), specific for (+)-anti-BPDE DNA adduct, was utilized for quantitative estimation of this bulky lesion in nanograms amounts of membrane transblotted DNA fragments. A carcinogen dose-dependent quantitative antibody binding response, due to selective recognition of the major (+)-anti-BPDE adduct, was seen with various DNA fragments separated by gel electrophoresis. The sensitivity of the immunodetection at 0.2 fmol (+)-anti-BPDE DNA adduct, allowed a linear detection in the range of modification level of 0.64 x 10(-7) to 86 x 10(-7) adducts per nucleotide in plasmid DNA. Based on this sensitivity, detection of 0.07 and 0.46 (+)-anti-BPDE DNA adducts in respective tk and vector DNA fragments was achieved upon immunoanalysis of the in vitro modified DNA. Adduct concentration dependent antibody binding was independent of size of the vector or insert fragments. Antibody binding response, to DNA modified in vivo, was dependent upon the dose of (+/-)-anti-BPDE to plasmid DNA replicating within bacterial hosts. The repair of (+)-anti-BPDE DNA adducts was determined as the loss of antibody binding sites in the specific fragments of plasmid DNA within host E. coli. About 50% of the initial DNA damage was repaired from the individual fragments during 15 min post-incubation in the repair-proficient (wild-type) E. coli cells. Complete adduct removal occurred in approx. 60 min of post-incubation period. A significant (91%) decrease in the survival of mutant (uvrA- recA-) cells was observed at 4 microM (+/-)-anti-BPDE treatment without any reduction in the colony forming units in the wild-type cells. On the contrary, no repair was seen in the excision repair-deficient (uvrA-) E. coli cells. The results indicate (1) the selectivity of the immunological method and the unique ability of the (+)-anti-BPDE specific antibodies to monitor the direct loss of this promutagenic base lesion from the in vivo modified DNA (2) the role of host excision repair pathway in efficient removal of adducts from bacterial genome determines the survival of the bacterial cells and (3) the repair of (+)-anti-BPDE DNA adducts in episomally replicating, transcriptionally active sequences occur at a rapid rate presumably due to the ease of accessibility of repair enzymes to lesions within DNA.

A sequence specific endonuclease from Micrococcus radiodurans.

A new sequence specific endonuclease, Mra I has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage lambda DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on phi X174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage lambda DNA are different from those cleaved by Sma I, Xma I and Xor II. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of lambda DNA. Mra I is shown to be an isoschizomer of Sac II and Sst II recognizing the palindromic nucleotide sequence '5-CCGC reduced GG-3'. The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45 degrees C and pH 7.0. Mra I represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.

USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability.

DNA polymerase eta (Polη) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Polη-proficient but not in Polη-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover.

Prognostic and aetiological relevance of 8-hydroxyguanosine in human breast carcinogenesis.

In order to estimate the level of oxidative damage and its role in breast cancer, the promutagenic oxidative lesion, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was determined in DNA isolated from 75 human breast tissue specimens and from normal and transformed human breast cell lines, utilising a newly developed solid-phase immunoslot blot assay. The amount of 8-OHdG was found to be 0.25 +/- 0.03 pmol/microgram in normal breast tissue from reduction mammoplasty, 0.98 +/- 0.174 pmol/microgram in benign tumours and 2.44 +/- 0.49 pmol/microgram DNA in malignant breast tissue with invasive ductal carcinoma. The malignant tissue had a statistically significant 9.76-fold higher level of 8-OHdG than normal tissue (P < 0.001, Mann-Whitney). A statistically significant 12.9-fold (P = 0.004) higher endogenous formation of 8-OHdG was also observed in cultured breast cancer cells compared with normal breast epithelial cells. In addition, a significantly elevated level (3.35-fold higher, P < 0.05) of 8-OHdG observed in oestrogen receptor-positive compared with oestrogen-negative malignant tissues, and in breast cancer cell lines (9.3-fold higher, P = 0.007) suggests a positive relationship between 8-OHdG formation and oestrogen responsiveness. The extent of 8-OHdG adducts did not show a discernible correlation with either the age or the smoking status of the patients. These results indicate that the accumulation of 8-OHdG in DNA has a predictive significance for breast cancer risk assessment and is conceivably a major contributor in the development of breast neoplasia.

Localization of O6-alkylguanine transferase in cancer susceptible cells of human female breast.

The cell type specific distribution of O6-alkylguanine-DNA-alkyltransferase (AGT) protein was assessed using immunohistochemical localization in human female breast tissue sections and biochemical quantitation of fractionated cell extracts. The results demonstrated that the AGT protein is predominantly localized in luminal epithelial and myoepithelial cells of the intralobular mammary ducts. Western blot analysis revealed that the AGT level in epithelial cell rich organoid fraction was substantially higher than the whole tissue and fibro-collagenous stromal cell fraction of the normal breast. The quantitative activity measurements confirmed the occurrence of a statistically significant 2.7-fold (P = 0.05) and 4.0-fold (P = 0.04) enriched AGT activity in extracts prepared from the organoids compared to the whole tissue homogenate and fractionated stromal cells, respectively. The results suggest that the invariably high AGT level in malignant compared to the normal breast tissue could be due to AGT accumulation in luminal epithelial and myoepithelial cell mass, increasing as a consequence of the uncontrolled proliferation and differentiation of ductal cells in invasive carcinoma.

In situ pyrimidine dimer determination by laser cytometry.

By using antiserum against pyrimidine dimers and argon-laser imaging microspectrofluorometry, we established a new method to determine UV-induced pyrimidine dimers and their repair in individual human cells. The method was sensitive enough to determine dimers induced by UV dose as low as 2 J/m2. Normal human cells repaired 50 and 60% of total damage within 8 and 24 h after UV irradiation (20 J/m2), but Xeroderma pigmentosum cells (complementation group A) were unable to repair any within the same period. Therefore, the method proved to be a quick, easy, sensitive and accurate means to determine pyrimidine dimers in situ.

Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage.

To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1-5 mM) and ENU (1-15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H-ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/microgram MNU alkylated and 1.6 to 0.6/microgram ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/MspI restriction length polymorphism (RFLP) at codon 12 of H-ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H-ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H-ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.

Site-specific induction and repair of benzo[a]pyrene diol epoxide DNA damage in human H-ras protooncogene as revealed by restriction cleavage inhibition.

Most genotoxic DNA base modifications localized at key genomic sequences constitute the molecular alterations crucial or mutagenesis and tumorigenesis. We have utilized lesion-rendered inhibition of restriction endonuclease cleavage for the analysis of site-specific DNA damage induced by (+/-)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, anti-BPDE) in human genes. The H-ras protooncogene and insulin gene sequences were used as targets for modification in vitro and in vivo. Selective induction of individual facultative bands, resulting from covalent modification of the cognate recognition sites, was observed in modified plasmid DNA for a number of restriction nucleases. The ras gene-specific damage, at the PstI, BstYI, NotI and BstEII recognition sites, was visualized and quantitated in human genomic DNA adducted by anti-BPDE. Repair of lesions at hexanucleotide sequences and/or regions surrounding the restriction site, was assessed as a gradual disappearance of facultative bands in DNA from repair-proficient human fibroblasts exposed to the carcinogen in confluent culture. Efficiency of the PstI site-specific repair was compared at low and high levels of initial damage. Higher genotoxic dose caused a decrease in the extent of adduct removal from the bulk DNA, while the specific site of the ras gene was still subject to fast repair. No measurable PstI site-specific repair was detected in the insulin gene. These results show the region-selective induction of bulky anti-BPDE DNA damage in non-related genomic targets and suggest that repair of these lesions in human cells proceeds with the efficiency tightly controlled at different levels of initial genotoxic load.